Biorad iCycler cfx manager files and HTqPCR
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Dear Sir/Madam, I would like to submit a question and in doing so am very grateful for your time, effort and expertise in doing so. I am just in the instruction phases of qPCR and using the Biorad iCylcer with cfx manager on a Dell optimplex 980, intel core i5, using R 2.15.0, with Windows XP prof 2002. The cfx files when exported into excel format look nothing like the example given from within the HTqPCR example file. The cfx exports both Ct and expression files, and includes all samples per page per plate. When trying to call in HTqPCR I get error code: " error in data.frame(sample = 1:length(samples), row.names = samples) : row names supplied are of the wrong length I performed qPCR on 2 plates for a total of 6 genes (5 target and 1 housekeeping), samples were 5 control mice, and 5 KI mice, the cfx manager file refers to numbers v655 etc as such per mouse. "Target 18s is housekeeping gene". HTqPCR program data file example: X1 Treatment A1 Passed Sample01 Gene1 Endogenous.Control X11.566983 X X.1 X.2 X.3 X.4 X.5 X.6 FALSE. 1 2 Treatment A2 Passed Sample01 Gene2 Target 34.9502 NA 23.11739 0.10150807 0 1 0.7232906 1.382570 FALSE 2 3 Treatment A3 Passed Sample01 Gene3 Target 27.962915 NA 16.17094 0.09178536 0 1 0.7460847 1.340330 FALSE 3 4 Treatment A4 Passed Sample01 Gene4 Target 29.946945 NA 18.09563 0.11069714 0 1 0.7023879 1.423715 FALSE 4 5 Treatment A5 Passed Sample01 Gene5 Target 26.987282 NA 15.13619 0.11057243 0 1 0.7026675 1.423148 FALSE 5 6 Treatment A6 Flagged Sample01 Gene6 Target 35.69901 NA 24.51676 0.61402905 0 1 0.1409204 7.096207 FALSE 6 7 Treatment A7 Passed Sample01 Gene7 Target 29.954237 NA 18.12556 0.09980855 0 1 0.7272241 1.375092 FALSE My cfx manager output file (Ct data only not expression file): Well Fluor Content Target Sample Threshold Cycle ( C(t) ) C(t) Mean C(t) Std. Dev A01 SYBR Unkn-01 18s v655 13.44 13.48 0.056 A02 SYBR Unkn-04 18s v656 14.22 14.22 0.000 A03 SYBR Unkn-07 18s v657 13.93 13.91 0.025 A04 SYBR Unkn-10 18s v658 13.92 13.88 0.048 A05 SYBR Unkn-13 18s v659 14.11 14.16 0.073 I realize most of my problem must be in my not comprehending what the top row in the HTqPCR file stands for (X, X.1, X2X3X4, etc) Questions: 1. How should I structure the cfx files once exported to excel. 2. I assume from HTqPCR manual that only one sample per file, so 10 files total (5 control mice vs 6 genes tested, 5 Ki mice with 6 genes tested). 3. Does a separate file need to be created for expression results? I understand many of you are incredibly busy and I am extremely grateful for and respect your time and effort. I apologize for what may seem simplistic questions. I have taken R classes but seems all the software my lab is using including microarray is not what was used when some of these program were built, and my inexperience. Sincerely, Brian Gorsuch [[alternative HTML version deleted]]
Microarray qPCR HTqPCR a4 Microarray qPCR HTqPCR a4 • 1.2k views

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